Artificial cartilage containing chondrocytes obtained from costal cartilage and preparation process thereof

ABSTRACT

The present invention relates to an artificial cartilage containing mesenchymal stem cell (MSC)-like dedifferentiated cells obtained by passage culturing costal chondrocytes, and a preparation process thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of copending application Ser. No.12/159,204 filed on Jun. 25, 2008, which is the National Phase under 35U.S.C. § 371 of International Application No. PCT/KR2006/004479, filedon Oct. 31, 2006, which claims the benefit under 35 U.S.C. § 119(a) toPatent Application No. 10-2005-0103156, filed in Korea on Oct. 31, 2005,all of which are hereby expressly incorporated by reference into thepresent application.

TECHNICAL FIELD

The present invention relates to an artificial cartilage containingchondrocytes obtained from costal cartilage and a preparation processthereof.

BACKGROUND ART

Damage to articular cartilage is an exceedingly common problem affectingthe joints of millions of people. The ability of adult cartilage toregenerate is limited due to avascularity and the absence of stem cellin this tissue. Although defects that extend to subchondral bone provokethe formation of a fibrous or fibrocartilagious tissue, it undergoespremature degeneration because the repair tissue is biochemically andbiomechanically different from hyaline cartilage.

A variety of clinical procedures have been developed to repair articularcartilage defects, but success has been limited. Bone marrow stimulationtechnique (fine joint plastic surgery and drill articular plasticsurgery) interpenetrate subchondral bone, and stimulate multipotent stemcells within bone marrow to repair the defects into a fibrous tissue orfibrocartilage. However, these methods have disadvantages that therepair fibrocartilage lacks such biochemical and biomechanical propertyas normal hyaline cartilage has. Further, their effects have not beenproved yet for the large area defects, osteoarthritis and the olderpatient. Osteochondral/chondral graft is simple and effective for smallsize defect, but if the tissue is moved from low weight part to highweight part, it may cause a harmful compression due to non-physiologicalbearing in the transplanted position. Periosteal and perichondrial grafthas a potential advantage to introduce new cell assembly which is ableto construct cartilage, but has also disadvantage that hyaline-likerepair tissue tends to undergo calcification through osteogenesis incartilage.

Furthermore, several cell-based procedures using chondrocytes,mesenchymal stem cells (MSCs), periosteocytes and perichondriocytes havebeen developed. Chondrogenic progenitor cells such as MSCs,periosteocytes and perichondriocytes get popularity more and more aspotential cell sources to repair the osteochondral defects. However, theprogenitor cells capacity to construct articular chondrocytes islimited, and the age of patient is directly related to clinical results.Further, it is reported that the repair hyaline-like tissue tends toundergo calcification.

Autologous articular chondrocyte transplantation (ACT) has beenclinically applied in small defects of articular cartilage, but onlylimited success has been reported. Even though articular chondrocytescan be easily isolated from mature articular cartilage by enzymedigestion, it requires two step-procedures for harvesting and graftingwhich are invasive to joint and considerably expensive. Thus ACT cannotbe applied to more than two small lesions, to the lesion size largerthan 10 cm², to the patient of rheumatoid or immune-related arthritis,and to the older patient due to articular cartilage degeneration withage.

Furthermore, only one to two percentage by the volume of articularcartilage is chondrocyte, in which approximately 2,000 cells/mg of humanarticular cartilage can be isolated for culture. An average 3 cm² defectrequires 9×10⁶ cells for the ACT procedure if implanted at celldensities similar to that found in the normal human knee joint. Inreality, more cells may be required for ACT since 26% of patients underthe age of 40 with grade IV chondromalacia lesions had multiple lesions.Therefore, a large number of chondrocytes are required to filladequately a volume of defect with a similar cell density as seen innormal human articular cartilage.

During serial monolayer culture for cell expansion, chondrocytes tend tostop expressing cartilage-specific proteoglycan and type II collagen,and switch to express type I collagen with producing a small amount ofproteoglycan. Such dedifferentiation is a main problem which limits cellexpansion in vitro and ACT application.

Further, costal cartilage is the biggest permanent hyaline cartilage inthe mammalian body, which has been suggested as a possible alternativedonor source for autologous graft in reconstruction of articularcartilage, external ear and trachea. Costal cartilage has been used torepair osteochondral defects in small joint such as the interphalangealjoints and the temporomandibular joints. Costal cartilage seems to haveseveral advantages over articular cartilage as donor tissue. Activelyproliferating chondrocytes were detected even in patients 80 years ofage or older, and a significant amount of costal cartilage is availablein patients younger than 60 years. In addition, costal cartilage isabundant, and its easy surgical accessibility allows less harm to donorsite. Therefore, if costal cartilage has the same phenotype as hyalinecartilage of articular cartilage, it can be considered the most usefulsource for treating a variety of articular cartilage disorders such asrheumatoid arthritis, osteoarthritis and cartilage defects of thejoints.

However, so far, only autologous tissue transplantation to graft costalcartilage per se to articular cartilage part has been performed, andthere has been no effort to isolate chondrocytes from costal cartilageto regenerate articular cartilage by tissue engineering method.

Furthermore, transplantation of chondrocytes or chondrogenic cells alonehas shown to be successful in rabbit models, but the healing rate haslimited due to loss of their viability in the transplanted cells and dueto the difficulty of fixing chondrocytes in the defect. To overcome thedifficulties related to the surgical procedure and to find a method ofmaintaining chondrocytes in the defect without outflow of the cells inthe articular cavity, new approaches in different biomaterial carriersas scaffolds onto which cells are seeded, have been studied. Idealscaffolds should be biocompatible, bioabsorbable or remodeled, andprovide framework that facilitates new tissue growth. They should alsodisplay material and mechanical properties compatible with articularcartilage function. A variety of biomaterials, naturally occurring suchas collagen-based biomaterial; collagen type I and II orcollagen/glycosaminoglycan (GAG) composites and synthetic such aspolyglycolic acid (PGA) and poly-lactic acid (PLLA), or their compositemixture, PLGA (poly D,L-lactic-co-glycolic acid), have been introducedas potential cell-carrier substances for cartilage repair. They haveshown that cartilage-specific extracellular matrix (ECM) components suchas type II collagen and GAG play a critical role in regulatingexpression of the chondrocytic phenotype and in supportingchondrogenesis both in vitro and in vivo.

Chitosan is the alkaline de-acetylated product of chitin and a family ofpoly-D-glucosamine units that vary in their degree of deacetylation andmolecular weight. Many investigators suggested that chitosan might beconsidered as a structural biomaterial for the repair of connectivetissues because of its structural similarity to GAGs found in theextracellular matrix. Chitosan and some of its degraded products can beinvolved in the synthesis of the articular liquid components such aschondroitine, chondroitine-sulfate, dermatane-sulfate, keratane-sulfateand hyaluronic acid (HA). These substances are necessary for nutritionof the cartilage. The fact that chitosan is polycationic, and itsstructure is similar to hyaluronic acid which is an important moleculeof ECM of articular cartilage has a particular importance for cartilagetissue engineering. Use of chitosan-based matrices in the tissueengineering of hyaline cartilage has been recently reviewed. Lahiji etal. (2000) showed that chondrocytes which are cultured on chitosan filmsmaintain differentiated phenotype and express cartilage specific ECMproteins such as type II collagen and sulfated proteoglycan. Studiesexploring the use of chitosan to potentiate neochondrogenesis have shownthe ability of chitosan to promote the maintenance of the chondrocytephenotype and biosynthesis of cartilage specific ECM components whengrown on chitosan films (Lahiji et al, 2000). Chitosan has also beenshown to potentiate the differentiation of osteoprogenitor cells and mayhave also enhanced bone formation.

Hyaluronic acid plays a vital role in many biological processes such astissue hydration, proteoglycan (PG) organization in the ECM, and celldifferentiation. It is also a component of healthy articular cartilage.Patti et al. (2001) showed that HA improved in vitro substrate adhesionability and proliferative activity of human cartilage cells. HA alsoimproved clinical function in early arthritis (Patti et al., 2001).

Further, fibroblast growth factor (FGF) is a strong mitogen forconnective tissue cells and MSCs (J. Cell Biol. 100, 477-485, 1985).During cell expansion, FGF inhibits the formation of thick F-actinstructure to maintain chondrogenic potential of articular chondrocytes(Exp. Cell Res. 253, 681-688, 1999). Also, FGF is known to maintainmultifamily differentiation of MSC throughout numerous mitoses.

SUMMARY OF THE INVENTION

First, the present inventors have studied whether chondrocytes obtainedfrom costal cartilage can be used as cell source for tissue engineeredartificial cartilage. Also, they have studied a way to solve the problemthat chondrocytes tend to lose chondrocytic phenotype because ofdedifferentiation during the passage. Further, they have continued toevaluate whether chitosan-based scaffolds onto which costal chondrocytesare seeded can be used for articular cartilage regeneration.

As a result, they discovered that chondrocytes obtained from costalcartilage are better than those from articular cartilage as donor cellsource for cartilage repair. Also, they discovered that dedifferentiatedchondrocytes during the passage show MSC properties to confirm theirability for use as cell therapeutic agent as well as artificialcartilage, by redifferentiating them into desired cells in thedifferentiation inducible medium. In addition, the present inventorsconfirmed that chondrocytes-loaded chitosan-based scaffolds whentransplanted to articular defects show effective articular regeneration,to complete the present invention.

Thus, the object of the present invention is to provide an artificialcartilage containing MSC-like dedifferentiated cells obtained bypassaging costal chondrocytes and a preparation process thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing comparison of in vitro cell expansion abilitybetween articular chondrocytes (ACs) and costal chondrocytes (CCs).

FIGS. 2 and 3 are pictures showing the morphological change and type IIcollagen expression of ACs and CCs at each passage.

FIG. 4 is a picture showing CCs morphology at P7, FIGS. 5 and 6 arepictures showing type I and II collagen and smooth muscle actin (SMA)antibody expression.

FIG. 7 is a graph showing cell expansion rate in DMEM-FBS, MSCGM andMSCGM-FGF.

FIGS. 8 and 9 are pictures showing type I collagen, type II collagen andSMA antibody expression of the cells cultured in DMEM-FBS, MSCGM andMSCGM-FGF, respectively.

FIG. 10 shows the results of Safranin-O staining for GAG to confirmdifferentiation of the cells cultured in DMEM-FBS, MSCGM and MSCGM-FGFinto chondrocytes.

FIG. 11 shows the results of redifferentiation of costal chondrocytes atP7 into cartilage in pellet form in cartilage differentiation medium.

FIG. 12 shows the results of Safranin-O staining for GAG to confirmdifferentiation into chondrocytes.

FIG. 13 is a picture showing Alkaline phosphatase expression asosteogenic differentiation initial marker in the cells which are inducedto be differentiated into osteoblasts, and FIG. 14 is a picture showingthe results of Alizarine red staining as osteogenic differentiationmarker.

FIG. 15 shows the results of Oil red O staining for lipid droplets inthe cells which are induced to be differentiated into adipocytes.

FIG. 16 is a schematic view showing the process for articular cartilagedefect repair by costal chondrocytes loaded chitosan-based scaffolds.

FIG. 17 is a graph showing comparison of chondrocytes growth rate inCol-GAG, CS and CS-HA by MIT assay.

FIG. 18 shows the results of the amount of PIP and GAG measured.

FIG. 19 shows cells morphology by H & E staining.

FIG. 20 shows GAG accumulation by Safranin-O staining and fast greenstaining.

FIGS. 21 and 22 show the results of immuno-staining for type I collagenand type II collagen, respectively.

FIG. 23 is a picture showing gross appearance of repair tissue ofarticular cartilage defect.

FIGS. 24 to 26 are pictures showing repair tissue of articular cartilagedefect by optical microscope.

FIG. 27 shows the results of Safranin-O staining for GAG distribution inrepair tissue of articular cartilage defect.

FIG. 28 shows the results of immuno-staining for type I and II collagenin repair tissue of articular carilage defect.

FIG. 29 is a macroscopic picture of the artificial cartilage fromMSC-like dedifferentiated cells loaded chitosan-based scaffolds inchondrogenic medium.

FIG. 30 shows the results of Safranin-O staining for GAG in chondrocytesredifferentiated in chondrogenic imdium.

FIG. 31 is a macroscopic picture of rabbit articular cartilage defect at6 weeks after transplantation with MSC-like dedifferentiated cellsloaded chitosan-based scaffolds.

DISCLOSURE OF THE INVENTION

First, the present invention relates to an artificial cartilagecontaining MSC-like dedifferentiated cells obtained by passaging costalchondrocytes. It is preferable that the costal chondrocytes are passagedin MSC growth medium (MSCGM), in particular fibroblast growth factor(FGF)-containing MSCGM for better cell expansion and differentiationinto cartilage.

Second, the present invention relates to an artificial cartilagecontaining redifferentiated chondrocytes obtained by culturing theMSC-like dedifferentiated cells in chondrogenic medium. In oneembodiment, the MSC-like dedifferentiated cells are pellet cultured inchondrogenic medium.

Third, the present invention relates to an artificial cartilage whereinchondrocytes are loaded on chitosan-based scaffold. The chitosan-basaedscaffold is preferably selected from the group consisting of chitosansponge; Transforming Growth Factor-β (TGF-β) containing chitosan sponge;hyaluronic acid (HA)-coated chitosan sponge; chondroitine-sulfate-coatedchitosan sponge; and chitosan-collagen composite sponge. Morepreferably, the chitosan-basaed scaffold is HA-coated chitosan sponge orHA-coated chitosan-collagen composite sponge.

Fourth, the present invention relates to a process for preparing anartificial cartilage comprising a step of passaging costal chondrocytesto obtain MSC-like dedifferentiated cells. In one embodiment, the costalchondrocytes are passaged in MSCGM or FGF-containing MSCGM. In anotherembodiment, the MSC-like dedifferentiated cells obtained by passagingcostal chondrocytes are redifferentiated by culturing them inchondrogenic medium, preferably by pellet culturing in chondrogenicmedium. In another embodiment, the process includes a step of loadingthe MSC-like dedifferentiated cells on chitosan-based scaffold.

Fifth, the present invention relates to a cell therapeutic agentcontaining MSC-like dedifferentiated cells obtained by passaging costalchondrocytes. In one embodiment, the cell therapeutic agent containsredifferentiated chondrocytes obtained by culturing the MSC-likededifferentiated cells in chondrogenic medium. In another embodiment,the cell therapeutic agent contains osteoblasts obtained by culturingthe MSC-like dedifferentiated cells in osteogenic medium. In anotherembodiment, the cell therapeutic agent contains adipocytes obtained byculturing the MSC-like dedifferentiated cells in adipogenic medium.

Below, the present invention is explained in more detail.

The main technical limitation in ACT for articular cartilage repair isthat a donor cartilage without weight-bearing in knee joint has low cellgrowth capacity, and it is difficult to obtain adequate number ofchondrocytes for covering cartilage defect according todedifferentiation of chondrocytes during in vitro cell expansion. Inaddition, success of ACT application is restricted by the patient ageand narrow choice of donor site.

Thus, in the present invention, it was first estimated whether costalcartilage which maintains a longer growth capacity during its existenceand has a larger donor tissue in human body can be applied as donor cellsource for ACT. First, based on initial cell yield, cell expansion rateand dedifferentiation, it was evaluated whether chondrocytes isolatedfrom costal cartilage can be applied as a potential cell source fortissue engineered articular cartilage. Particularly, in the presentinvention, initial cell yield and cell expansion rate in monolayerculture of chondrocytes obtained from costal cartilage are compared tothose of chondrocytes from articular cartilage. In addition,dedifferentiation rate during in vitro cell culture is estimated by cellmorphology and type II collagen expression.

As a result, costal cartilage gave 2.6 folds higher initial cell yieldthan articular cartilage. During in vitro cell culture, costalchondrocytes (CCs) more rapidly grew, and gave about 3.0 folds highercell expansion up to P4 than articular chondrocytes (ACs). During serialculture, ACs and CCs gradually lost their chondrocytic phenotype butinstead converted into fibroblast-like cells, and type II collagenexpression decreased. The loss of their original phenotype was moreexpedited in CCs than ACs. Comparing the same number of passage, CCsmore rapidly dedifferentiated than ACs.

The results of first culture represented by comparison of ACs and CCsfrom the same rabbit which have dedifferentiation and growth profileaccording to in vitro cell expansion passage suggest that costalcartilage can be used as potential cell source for osteoarthritisrepair.

In further study, the present inventors confirmed that dedifferentiatedcells obtained by passaging costal cartilage have MSC properties. MSC ischondrogenic progenitor cells, and has a meaning as potential cellsource for repairing osteochondral defect. Thus, in the presentinvention, it was first disclosed that MSC-like dedifferentiated cellsobtained from costal chondrocytes are chondrogenic progenitor cellswhich can be used for osteochondral defect repair.

MSC-like dedifferentiated cells according to the present invention wereable to differentiate into osteoblasts and adipocytes as well aschondrocytes. Thus, in the present invention, the term “MSC-likededifferentiated cells” refers to cells which are dedifferentiated bypassage and have potential to differentiate into chondrocytes,osteoblasts, adipocytes, etc., like MSC. That is, the term refers tomultipotent cells.

In further study, the present inventors disclosed that when costalchondrocytes are passaged in, in particular FGF-containing MSCGM, thecell expansion rate is outstanding, and differentiation intochondrocytes is excellent. It is anticipated that normal cell culturemedium containing DMEM may have the same result if FGF is added thereto.

Furthermore, in the present invention, as scaffold for loadingchondrocytes, chitosan-based scaffold, in particular sponge formHA-coated chitosan scaffold was used to estimate the role of culturedautologous costal chondrocytes laden HA-coated chitoan compositescaffold in the repair of full-thickness articular cartilage defects ina weight-bearing site in animal model. The use of the rabbit knee modelin assessment of cartilage repair has been widely used, and spontaneoushealing of full-thickness 3 mm-diameter chondral defects in the rabbitpatellar groove has been reported to occur within 6 to 8 weeks in 4 to 6months old rabbit. Thus, this period is needed to reveal mostdegenerative failures of the apparently healed carilage. In the designedanimal model experiment as below (4 mm diameter osteochondral defect),it was shown that autologous costal chondrocytes within chitosan-basedscaffold according to the present invention very effectively repairedfull-thickness articular cartilage defects in a weight-bearing site.

Hereinafter, the present invention will be described in more detail withreference to the following Examples, but the scope of the presentinvention should not be construed to be limited thereby in any manner.

EXAMPLES Example 1: Evaluation on Whether Costal Chondrocytes can beDonor Cell Source for Articular Cartilage Repair Materials and Methods

Isolation of Chondrocytes

Articular and costal cartilages were obtained from 3 to 4 months old NewZealand White Rabbit, and then weighed. To harvest costal cartilage,animals were anesthetized by intravenous injection of a mixture ofxylazine hydrochloride (2 mg/weight kg, Rompun, Bayer, Korea) andketamine hydrochloride (6-10 mg/weight kg, ketalar, Yuhan Co., Korea).The skin in the region of the chest was shaved, washed with alcohol, andprepared with povidone-iodine solution. The back side of the right chestwas opened, and 9^(th) and 10^(th) costal cartilages were exposed. Thecostal cartilages, after removing soft adhering tissues, were thenweighed. These cartilages were minced into 1-2 mm³ pieces and rinsed 3times in D-PBS (Dulbecco's phosphate buffered saline; Jeil Biotechservices Inc., Taegu, Korea). After being rinsed, the minced cartilagewas digested by the enzyme cocktail solution including collagenase D (2mg/ml, Roche Diagnostic GmbH, Germany), hyaluronidase (1 mg/ml, Roche),and DNase (0.75 mg/ml, Roche) under 37° C. and 5% CO₂ for overnight.Then the solution was filtered through a 53 μm nylon mesh, isolatedcells were washed 2 times with DMEM (Dulbecco's Modified Eagle Medium;Gibco Life Technologies, Grand Island, N.Y., U.S.A.) supplemented with10% fetal bovine serum (FBS; Hyclone technologies, U.S.A.) and 1%penicilline/Streptomycin/Fungizone cocktail (Gibco), and viable cellswere counted on Haemocytometer based on the trypan blue exclusion. Thecells were plated at a cell density of 5×10⁵ cells/100 mm diameter Petridish, the culture medium was changed every other day, and fresh 50 μg/mlL-ascorbic acid (Sigma, St. Louis, Mo., U.S.A.) was added at each mediumchange. The primary cells at confluence were subcultured up to P4.

MTT Assay

For MTT assay, ACs and CCs at each passage were seeded at cell densityof 1×10⁵ cells per well of 6-well culture plate and cultured for 5 days.The cell growth was determined based on MTT assay (Mosman T. Rapidcolormetric assay for cellular growth and survival, application toproliferation and cytotoxicity assays, J. Immunol. Methods 1983; 65:55-63). For the calibration of cell number from optical density (O.D.),standard curve was measured in the range of 1 to 8×10⁵ cells and O.D.was transformed to the cell number.

Immunofluorescence Staining of Type II Collagen

For immunofluorescence staining, the chondrocytes from each passage wereplated onto cover slip, cultured for 2 days, fixed with 3.7% formalin inPBS for 10 minutes, and permeabilized with 0.2% Triton X-100 in PBS. Thepermeabilized cells were incubated with 20% normal goat serum to blocknonspecific reaction and a monoclonal anti-type II collagen antibody(monoclonal anti-mouse antibody; Chemicon international Inc., Temecula,Calif., U.S.A.) was used as a primary antibody. Fluoresceinisothiocyanate (FITC) labeled goat anti-mouse IgG conjugate (VectorLab., Burlingame, Calif., U.S.A.) was used as a secondary antibody, andthe cover slip was incubated for 5 minutes with4′,6-diamidino-2-phenylindole (DAPI; 1 g/ml; Sigma Chemical Co., St.Louis, Mo., U.S.A.) for nuclear staining. Cells were observed underfluorescence microscope (Olympus Optical Co., Japan).

Statistics

Statistical analysis was carried out by the unpaired t test to determinewhether variables were significantly different (p<0.05) in the eachexperiment.

Results

Comparison of Initial Cell Yield and Cell Expansion Rate of ChondrocytesIsolated from Articular Cartilage and Costal Cartilage

In order to search potential donor site suitable for autologouschondrocytes transplantation in adult, the cell yield of chondrocytesisolated from articular cartilage and costal cartilage are compared(Table 1).

TABLE 1 Comparison of initial cell yield of chondrocytes isolated fromarticular cartilage and costal cartilage 1 2 3 Mean (cells/mg) ± S.D. AC6,000 7,000 13,400  8,800 ± 3,278^(a) CC 20,000 26,600 22,100 22,900 ±2,753 ^(a)Mean value ± S.D. from three independent cell isolations.Statistical analysis was carried out by the student t-test (p < 0.05).

As shown in Table 1 above, the articular cartilage and the costalcartilage gave initial cell yields of 8,800±3,278 cells/mg and22,900±2,753 cells/mg, respectively, which corresponds approximately 2.6folds higher cell yield in the costal cartilage than in the articularcartilage. Thus, costal cartilage seems to be the better donor site forautologous chondrcocytes than articular cartilage in terms of initialcell yield.

Then, in vitro expandability of chondrocytes from articular cartilage orcostal cartilage was compared. The cells from each passage wereseparately plated at a density of 1×10⁵ cells per well of 6-well plateand MTT assay was performed at 5th day after seeding (FIG. 1).Throughout the cell passages, overall growth rates of CCs were higherthan those of ACs. At P0, the growth rate of CCs was approximately twicethan that of ACs. At P2, the growth rate of ACs was similar to that ofCCs, but at P3, the growth rate decreased than P2.

For the estimation of expandability of chondrocytes isolated fromarticular cartilage and costal cartilage, the time to reach confluenceand total cell number at each cell passage were compared after seedingthe same cell numbers (Table 2).

TABLE 2 Comparison of time to reach confluence and total cell expansionrate at confluence between ACs and CCs P0 to P4 P0 to P2 Expansion Time(days) Expansion rate (folds) Time (days) rate (folds) AC 20 ± 7^(a) 22± 8.4 37 ± 7.3 106 ± 22 CC 13 ± 3 54 ± 14.5 29 ± 3.4 310 ± 70 ^(a)Meanvalue ± S.D. from three independent experiments.

As shown in Table 2 above, in the aspect of the cell expansion advantagein vitro, CCs seem the better donor chondrocytes than ACs.

Comparison of the Loss of Chondrocytic Phenotype of ArticularChondrocytes with Costal Chondrocytes during in vitro Expansion

During in vitro cell expansion, chondrocytes frequently lose their invivo properties such as round shape, type II collagen and GAGexpression. Both morphological changes and expression of type IIcollagen were examined at each passage of ACs and CCs (FIGS. 2 and 3).From P2, some group of cells began to lose their original round butinstead converted into fibroblastic spindle shape (FIG. 2). At P4, mostCCs actually changed their morphologies into fibroblastic phenotype.However, this morphological change was much slower in ACs than CCs.Thus, ACs more slowly expand in vitro than CCs, and more slowly changestheir morphology during in vitro cell expansion than CCs.

For further confirmation on whether fibroblastic morphological change isaccompanied by the loss of type II collagen expression,immunofluorescence staining was performed with anti-type II collagenantibody (FIG. 3). At P0, 99% cells of primary chondrocytes derivedeither from costal cartilage or articular cartilage expressed type IIcollagen. At P2, the number of chondrocytes expressing type II collagenrapidly decreased, and this was much severer in CCs. At P4, most CCs didnot express type II collagen, whereas some of ACs maintained expressingtype II collagen.

The number of type II collagen expressing cells was counted among DAPIpositive chondrocytes (Table 3).

TABLE 3 Number of type II collagen expressing cells at different cellpassage (%) AC (%) CC (%) P0 98.6 ± 1.1^(a) 97.2 ± 2.4 P1 53.5 ± 7.837.6 ± 5.4 P2 23.6 ± 4.6 10.0 ± 3.3 P3 14.3 ± 3.0  5.1 ± 0.7 P4 10.4 ±2.8  1.0 ± 0.4 ^(a)Mean value ± S.D.

to As shown in Table 3 above, chondrocytes gradually lost their originalphenotype such as round cell morphology and type II collagen expressioncapacity during in vitro expansion, which was much faster in CCs thanACs.

Example 2: Confirmation on Whether Fully Dedifferentiated CostalChondrocytes Show MSC's Properties Materials and Methods

Isolation of Chondrocytes

Costal cartilages were obtained from 4 to 5 months old New Zealand WhiteRabbit. The skin in the region of the chest was shaved, washed withalcohol, and prepared with povidone-iodine solution. The back side ofthe right chest was opened, and 9^(th) and 10^(th) costal cartilageswere exposed. The costal cartilages, after removing soft adheringtissues, were then weighed. These cartilages were minced into 1-2 mm³pieces and rinsed 3 times in D-PBS (Dulbecco's phosphate bufferedsaline; Jeil Biotech services Inc., Taegu, Korea). After being rinsed,the minced cartilage was digested by the enzyme cocktail solutionincluding collagenase D (2 mg/ml, Roche Diagnostic GmbH, Germany),hyaluronidase (1 mg/ml, Roche), and DNase (0.75 mg/ml, Roche) under 37°C. and 5% CO₂ for overnight. Then the solution was filtered through a 53μm nylon mesh, isolated cells were washed 2 times with DMEM (Dulbecco'sModified Eagle Medium; Gibco Life Technologies, Grand Island, N.Y.,U.S.A.) supplemented with 10% fetal bovine serum (FBS; Hyclonetechnologies, U.S.A.) and 1% penicilline/Streptomycin/Fungizone cocktail(Gibco), and viable cells were counted on Haemocytometer based on thetrypan blue exclusion. The cells were plated at a cell density of 5×10⁵cells/100 mm diameter Petri dish, the culture medium was changed everyother day, and fresh 50 μg/ml L-ascorbic acid (Sigma, St. Louis, Mo.,U.S.A.) was added at each medium change. The primary cells at confluencewere subcultured up to P7.

Expression of Type I, II Collagen and Smooth Muscle Actin (SMA) Antibody

The costal chondrocytes at P7 were plated onto cover slip, cultured for2 days, fixed with 3.7% formalin in PBS for 10 minutes, andpermeabilized with 0.2% Triton X-100 in PBS. Anti-type I collagenantibody (polyclonal anti-goat antibody: southern), anti-type IIcollagen antibody (monoclonal anti-mouse antibody: Chemiconinternational Inc., Temecular, U.S.A.) or anti SMA antibody (monoclonalanti-mouse antibody: DAKO) were applied to the specimen for overnight at4° C., incubated with secondary antibody, then incubated withstreptavidin-peroxidase for 1 hour, and developed with 0.1%3,3′-diaminobenzidine tetrahydrochloride (DAB; Chromogen, DAKO Corp.,Calif., U.S.A.) in PBS for 5 minutes. Fast red dye (Vector Lab.,Burlingame, Calif., U.S.A.) was used for counterstaining.

Results

Confirmation on Dedifferentiation of Costal Chondrocytes and Expressionof MSC's Properties

As shown in FIG. 4, costal chondrocytes at P7 lost their original roundshape but instead converted into fibroblastic spindle shape. This showsthat costal chondrocytes fully dedifferentiated. FIGS. 5 and 6 show typeI, II collagen and SMA antibody expression in dedifferentiatedchondrocytes. In every cell, type I collagen is still expressed, buttype II collagen is not expressed at all. One of the characteristics ofMSC, expression of SMA was observed in many cells (FIG. 6). As above,the expression of type I collagen and SMA confirmed that passaged costalchondrocytes show MSC's properties.

Example 3: Evaluation on Expansion of Costal Chondrocytes and theirDifferentiation into Cartilage According to Culture Condition (DMEM+10%FBSs, MSCGM or FGF-Containing MSCGM) Materials and Methods

Isolation and Culture of Chondrocytes

Chondrocytes were isolated by the method as described above, and viablecells were counted on Haemocytometer based on the trypan blue exclusion.The chondrocytes were plated at a cell density of 5×10⁵ cells/100 mmdiameter Petri dish, and cultured in DMEM (DMEM-FBS), MSCGM (Cambrex BioScience Walkersville, Inc., MD, U.S.A.), or MSCGM added with 1 ng/ml ofFGF (R&D System Inc., MN, U.S.A.).

Cell Expansion Rate According to the Medium

When the dish was filled with the cells, the cells were removed byTrypsin-EDTA (Gibco), and viable cells were counted on Haemocytometerbased on the trypan blue exclusion to evaluate cell expansion rate.Then, the cells were plated at a cell density of 5×10⁵ cells/100 mmdiameter Petri dish, and cultured in the same medium.

Expression of Type I and II Collagen and SMA Antibody

The costal chondrocytes cultured in DMEM-FBS, MSCGM or MSCGM-FGF at eachpassage were plated onto cover slip, cultured for 2 days, fixed with3.7% formalin in PBS for 10 minutes, and permeabilized with 0.2% TritonX-100 in PBS. The permeabilized cells were incubated with 20% normalgoat serum to block nonspecific reaction, and anti SMA antibody(monoclonal anti-mouse antibody: DAKO) was used as a primary antibodyand applied to the specimen for overnight at 4° C. Fluoresceinisothiocyanate (FITC) labeled goat anti-mouse IgG conjugate was used asa secondary antibody, and the cover slip was incubated for 5 minuteswith 4′,6-diamidino-2-phenylindole (DAPI; 1 g/ml; Sigma Chemical Co.,St. Louis, Mo., U.S.A.) for nuclear staining. Cells were observed underfluorescence microscope (Olympus Optical Co., Japan).

After washing the bottom of the Petri dish 2 times with PBS, the costalchondrocytes cultured in DMEM-FBS, MSCGM or MSCGM-FGF at each passagewere dissolved with cell dissolving buffer (Biolabs; 20 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodiumpyrophosphate, 1 mM Na₃VO₄, 1 mM beta-glycerophosphate, 1 ug/mlleupeptin) added with 2 mM of phenylmethylsulfonylfluoride (PMSF) forinhibiting protein degradation enzyme activity, and centrifuged toobtain the supernatant for the use for collagen isolation. The proteinswere loaded to 10% SDS-PAGE, transferred into membrane, and performedwestern blotting with anti-type I collagen antibody and anti-type IIcollagen antibody (polyclonal anti-goat antibody: southern).

Differentiation Induction into Chondrocytes

The costal chondrocytes expanded up to P7 or P8 in DMEM-FBS, MSCGM orMSCGM-FGF were made into pellet, and induced to differentiate intocartilage in chondrogenic medium for 3 weeks. Safranin-O staining wasperformed to estimate the degree of differentiation into cartilage.

Results

Cell Expansion Rate Depending on the Medium

When the cells filled the dish, the cells were removed by Trypsin-EDTA(Gibco), and viable cells were counted on Haemocytometer based on thetrypan blue exclusion to evaluate cell expansion rate.

As shown in FIG. 7, to arrive up to P7, it took about 30 days inMSCGM-FGF wherein the cells expanded about 10⁷ folds, and it took about38 days in MSCGM where the cells expanded about 10⁵ folds. However, inDMEM-FBS, in about a month, cell expansion was hardly occurred withshowing a graph in parabolic shape, and at P7, the cell expanded about10³ folds only.

Thus, in the culture of costal chondrocytes, the expansion rate ofcostal chondrocytes was better in MSCGM than in DMEM+10% FBS. Inaddition, when cultured in MSCGM added with FGF, the expansion rate ofcostal chondrocytes was outstandingly superior. It can be anticipatedthat the same result would be obtained if the costal chondrocytes arecultured in DMEM added with FGF.

Expression of Type I and II Collagen and Anti-SMA Antibody

The expression degree of type I and II collagen and anti-SMA antibodywas measured for the costal chondrocytes cultured in DMEM-FBS, MSCGM orMSCGM-FGF.

As shown in FIG. 8, type II collagen expression decreased as the passageincrease, and the decreasing rate of type II collagen expression bycostal chondrocytes was higher in the order of MSCGM-FGF, MSCGM andDMEM-FBS.

In costal chondrocytes expanded in MSCGM and DMEM-FBS, the SMAexpressing cells were about 90%, namely, many cells expressed SMA.However, in costal chondrocytes expanded in MSCGM-FGF, very small numberof cells expressed SMA over passages.

As shown in FIG. 9, in costal chondrocytes expanded in DMEM-FBS andMSCGM, the type I collagen expression increased as the passage increase,and at P7, almost all the cells expressed type I collagen. In contrast,only 60 to 70% of the cells expanded in MSCGM-FGF expressed type Icollagen.

Expression of GAG

As shown in FIG. 10, the costal chondrocytes expanded in DMEM-FBS showGAG expression mainly at the outer wall of the pellet, and most cells inthe outer wall show chondrocytic morphology in the high poweredmicroscopic picture. The costal chondrocytes expanded in MSCGM show lowGAG expression in some of the pellet surface after 3 weeks cartilagedifferentiation, and very small number of cells shows chondrocyticmorphology. In contrast, the cells expanded in MSCGM-FGF show strong GAGexpression in most pellets after 3 weeks cartilage differentiation, andshow chodrocytic morphology.

Consequently, when MSCGM added with 1 ng/ml of FGF is used as culturemedium for expanding costal chondrocytes, chondrocytic differentiationcapacity is maintained after cell expansion of about 2×10⁷ folds.

Example 4: Redifferentiation of Fully Dedifferentiated CostalChondrocytes Materials and Methods

Isolation of Chondrocytes

The dedifferentiated cells cultured up to P7 in the Example 2 were used.

Differentiation of Fully Dedifferentiated Costal Chondrocytes intoChondrocytes

For redifferentiation of the fully dedifferentiated costal chondrocytesinto chondrocytes, the cells were inoculated 1×10⁶ cells/15 ml tube, andpellet cultured. The medium was changed to chondrogenic medium (Highglucose DMEM, 1% ITS+3 (Sigma), 100 nM dexamethasone (Sigma), 50 ug/mlvit C (Sigma), 40 ug/ml praline (Sigma), 10 ng/ml TGF β₃ (R&D system,Inc., MN, U.S.A.), and the control group was filled with basal medium(DMEM-HG, 10% FBS). The medium was changed 2 times a week, and observedat 2 and 3 weeks.

To confirm differentiation into chondrocytes, at 2 and 3 weeks, 3-Dconstructs were fixed with 3.7% phosphate-buffered formalin, prepared toparaffin specimen, and sectioned by 5 μm thickness. The thin sectionswere deparaffinized and stained with Safranin-O and fast green (Sigma)for observing GAG distribution.

Differentiation of Fully Dedifferentiated Costal Chondrocytes intoOsteoblasts

For redifferentiation into osteoblasts, the costal chondrocytes at P7were inoculated at a cell density of 2×10⁴ cells/cm² 6 well and 24 well,and cultured in osteogenic medium (DMEM, 10% FBS, 100 nM dexamethasone(Sigma), 10mM β-glycerol phosphate (Sigma), 50 ug/ml vit C (Sigma)). Themedium was changed 2 times a week, and observed at 2 and 3 weeks. Thebasal medium (DMEM, 10% FBS) was used for the control group.

To confirm differentiation into osteoblasts, the cells were stained withAlkaline phosphatase as differentiation initial marker. The degree ofexpression was histochemically observed with Alkaline phosphatasemeasurement kit (SIGMA-ALDRICH, St. Louis, Mo., U.S.A.). Also, the cellswere stained with Alizarin Red S (SIGMA-ALDRICH) which stains mineralcomponents secreted during osteoblasts maturation, to observe the degreeof differentiation into osteoblasts.

Differentiation of Fully Dedifferentiated Costal Chondrocytes intoAdipocytes

For redifferentiation into adipocytes, the costal chondrocytes at P7were inoculated at a cell density of 2×10⁴ cells/cm² 6 well and 24 well,and cultured in adipogenic medium (High glucose DMEM (Gibco), 10% FBS,10 mg/ml insulin (Sigma), 100 nM dexamethasone (Sigma), 0.2 mMindomethacin (Wako Pure Chemical Industries, Japan), 500 uM3-isobutyl-1-methylxanthin (Wako)) for 3 days, and repeated with 1 dayculture in adipogenic medium (High glucose DMEM, 10% FBS). The basalmedium (DMEM-HG, 10% FBS) was used for the control group, and observedat 2 and 3 weeks.

To confirm differentiation into adipocytes, the cells were stained withOil Red O which particularly stains lipid droplets.

Results

Differentiation into Chondrocytes

FIG. 11 shows a macroscopic picture of the results at 3 weeks ofdifferentiation induction of costal chondrocytes at P7 intochondrocytes. The cells one another formed the pellet, and the size ofthe pellet was bigger in cartilage induction group compared to thecontrol group (basal medium: DMEM, 10% FBS).

FIG. 12 shows the results of Safranin-O staining for GAG. At 3 weeks ofdifferentiation induction, GAG expression was observed in the outer wallof the pellet. The cells show chondrocytic morphology. The control groupdid not express GAG, nor show chondrocytic morphology.

Differentiation into Osteoblasts

To confirm differentiation into osteoblasts, Alkaline phosphataseactivity was observed. FIG. 13 is a picture showing Alkaline phosphatasemeasurement by using Alkaline phosphatase measurement kit(SIGMA-ALDRICH, St. Louis, Mo., U.S.A.). Alkaline phosphatase activity,as osteogenic differentiation initial marker, strongly expressed at 2weeks of differentiation, and tends to decrease a little at 3 weeks. Thecontrol group cultured in basal medium did not express Alkalinephosphatase activity.

Further, FIG. 14 show the result of mineral staining with Alizarin redstaining. No expression was observed at 2 weeks of differentiation, butpartial staining at 3 weeks to confirm maturation differentiation intoosteoblasts.

Differentiation into Adipocytes

To confirm differentiation into adipocytes, the cells were stained withOil Red 0 which particularly stains lipid droplets. As shown in FIG. 15,a number of lipid droplets stained with Oil Red 0 within the cellsemerged to confirm differentiation into adipocytes.

Example 5: Evaluation of Repair Effect of Costal Chondrocytes withinChitosan-based Scaffold on Articular Cartilage Defects Materials andMethods

The procedure of Example 5 is the same as the schematic view of FIG. 16.FIG. 16 shows the procedure where costal chondrocytes are isolated,cultured up to P2, and dedifferentiated cells are seeded ontochitosan-based scaffold, cultured, and the scaffold is transplanted intoarticular cartilage defect to repair cartilage defect.

Isolation of Chondrocytes

After taking the same procedure as the Example 1 above, the primarycells at confluence were subcultured up to P2.

Preparation of Sponge-form Scaffolds

COL-GAG Sponge:

The porous collagen matrix (Integra®, Integra Lifesciences Co., U.S.A.)was made of cross-linked bovine tendon collagen which is prepared tohave a controlled porousness and predetermined degradation rate, and GAG(chondroitin-6-sulfate) fiber. The scaffold (COL-GAG) was made by 4 mmbiopsy punch (S.F.M., Germany) and the silicon layer was removed by fineforceps.

CS Sponge:

Chitosan (Korea chitosan Co., Korea, 1.5% w/v) was dissolved in 0.1%aqueous acetic acid. After complete dissolution with sufficientagitation, this solution was passed 0.4 μm membrane filter (MilliporeCo., U.S.A.) and was shared into molds. The casted gels were placed at−80° C. for 24 hours and then freeze-dried in a lyophilizer for 24hours. The scaffolds were washed residue acid with alcohol and distilledwater, and added acetic anhydride to acetylation. The scaffolds werewashed sub-produced-acid with alcohol and distilled water and werefreeze-dried. Thickness of chitosan scaffold (CS) was approximately 2mm. Fine structure and pore size of chitosan scaffold were obtained bythe scanning electron microscopy (SEM; FIG. 8). The scaffold having 4 mmdiameter was made by 4 mm biopsy punch. Prior to the cell cultureexperiments, the scaffolds (CS) were sterilized by exposure toultraviolet light for 30 minutes and were soaked with culture medium for10 minutes. The prepared scaffolds were immediately used.

CS-HA Sponge:

After making 4 mm in diameter of chitosan scaffolds, they were soakedwith 50% ethanol and were re-soaked with 0.1% hyaluronic acid (HA;Sigma, St Louis, Mo., U.S.A.) in PBS. The scaffolds were placed at −80°C. for 24hours and then freeze-dried in a lyophilizer for 24 hours.Prior to the cell culture experiments, the scaffolds (CS-HA) weresterilized by exposure to ultraviolet light for 30 minutes and weresoaked with culture medium for 10 minutes. The prepared scaffolds wereimmediately used.

Chondrocytes Seeding onto Scaffolds and Culturing

COL-GAG, CS and CS-HA (4 mm in diameter×2 mm in thickness) were seededwith 2×10⁶ costal chondrocytes of second passage in medium volume of 20μl, which had been placed in 6-well plates at 37° C. with 5% CO₂ for 2hours. Subsequently, 6 ml of culture medium were added to each well andthe cells were cultured for 4 weeks. The medium was changed every weekand fresh L-ascorbic acid was added to the medium throughout the cultureperiod every 72 hours. The culture soup and cell-scaffold constructswere obtained at 2, 7, 14 and 28 days, and the constructs only wereobtained at 2 days.

MTT Assay, Blyscan Assay, Histological and Immunohistochemical Stainingof the Cell-scaffold Constructs, and Procollagen Type I C-peptide (PIP)ELISA Assay

MTT Assay

The cell proliferation rates within each scaffolds were determined basedon MTT assay (Mosman T. Rapid colormetric assay for cellular growth andsurvival, application to proliferation and cytotoxicity assays, J.Immunol. Methods 1983; 65: 55-63).

Procollagen Type I C-peptide (PIP) ELISA Assay

For quantitative determination of pro-C-peptide release from newlysynthesized collagen, pro-collagen type I C-peptide (PIP) EIA Kit(Takara Bio. Inc., Japan) was used. The culture medium was diluted to1:100 with distilled water and the rest of procedure was followedaccording to the manufacturer's specification. The absorbance wasmeasured at 450 nm with a microplate reader (Bio Rad Lab., Richimond,Calif., U.S.A.). The amount of released peptide was calibrated with thestandard curve in the range of 0-640 ng PIP/ml.

Blyscan Assay

The GAG content was determined quantitatively using a 1,9-dimethylmethylene blue assay (Blyscan® Glycosaminoglycan Assay, Biocolor, U.K.).Aliquot 300 μl of the culture medium was analyzed according to themanufacturer's specification and calibrated with standard curve measuredby 0.1, 0.2, 0.3, 0.5, 1 and 2, μg of shark chondroitin sulfate (SigmaChemical Co. St Louis, U.S.A.).

Statistics

Statistical analysis was carried out by the unpaired t test to determinewhether variables were significantly different (p<0.05) in eachexperiment.

Animal Care

24 New Zealand white rabbits (initial weight 2.2 to 2.5 kg, about 4 to 5months old) were used. Animals were regarded as young adults sinceperpendicular skeletal growth does not generally happen after 4 monthsold (Masoud et al., 1986). Animals were obtained 1 to 2 weeks before theexperiments, and maintained under fixed light and darkness period (7:00AM-7:00 PM, light period) and at fixed temperature of 22±2° C. andmoisture of 50±7%. Animals were freely provided with water and food(standard lab digest (pellet type, Purina Co., Korea)), and were allowedto move freely in the ground. Animals were housed, supervised, andhandled according to the approved national guidelines for animal care.

Surgical Procedure

Rabbits were anesthetized as described in the Example 1 above.Additional anesthesia was intravenous injection of a mixture of xyalzineand ketamine. Prior to operation, animals were intramuscularlyadministered with antibiotics (cefazoline, Jonggun-dang, Seoul, Korea)once.

The skin in the region of the knee was shaved, washed with alcohol, andprepared with povidone-iodine solution. The surgical approach wassimilar to that described by Shapiro et al. After a medial parapatellarincision was made, the patella was dislocated laterally to expose thepatella groove of the femur. A defect 4 mm in diameter was created inthe weight-bearing area of the patello-femoral joint by using alow-speed drill. This area is intermittently weight-bearing in therabbit and contacts the patella bone. The conical full-thickness defectsextended from the surface of the articular cartilage through the compactsubchondral bone into the cancellous bone in the marrow space of distalfemur. The depth of the defects was approximately 2.0 to 2.5 mm. Afterdrilling, the defect was irrigated extensively with cold saline (NaCl0.9% w/v) to remove loose fragments. A drop of a fibrin adhesive system(Tisseel® Kit, Baxter AG, Austria) was applied into the defects to fixthe transplanted construct in the defect and immediately scaffold wasinserted into the defects. In the control defects, only fibrin sealantwas used (FIG. 16). The dislocation of patella was reduced and the jointcapsule and skin were sutured in layers. Any cast was not applied andthe rabbits were allowed to move freely in their field immediately afterrecovery from anesthesia. Intramuscular injections of cefazoline wereperformed twice a day for 5 days. Knees of animals were separated intothree groups: (1) untreated defects (Control); (2) treated with CS-HAalone (S); or (3) treated with costal chondrocytes-laden CS-HA (S-CELL).At 6 and 12 weeks from the implant, rabbits were euthanized with aninjection of MgSO4 under deep anesthesia.

Histological Evaluation for Repair Tissues

After the sacrifice of the animals, their knees were examined for thegross morphology (color, integrity, contour and smoothness), repair ofthe defects and appearance of the surrounding cartilage. The specimensof the implantation areas were dissected, photographed and fixated with10% buffered formalin for a week at room temperature. Samples were thenrinsed to eliminate the excess fixative and decalcified by Calci-ClearRapid (National Diagnotics, U.S.A.) for a week. They were dehydrated bygrading ethanol and xylene and embedded in paraffin. 6 μm thick sectionswere cut by a microtome for histological studies. Sections were stainedwith hematoxylin and eosin (H & E) for the study of morphologic detailand with Safranin-O and fast green to assess GAG distribution.

Evaluation of Cartilage Defects

The sections were evaluated blindly by two investigators using amodified histological grading scale from those of Pineda et al (1992)and Wakitani et al (1994). The grading scale was totally composed of 5categories with a total range of points from 0 (normal cartilage) to 14(no repair tissue) (Table 4).

TABLE 4 Histological grading scale for the defects of cartilage CategoryPoints Cell morphology Hyaline cartilage 0 Mostly hyaline cartilage 1Mostly fibrocartilage 2 Mostly non-cartilage 3 Non-cartilage only 4Matrix staining (metachromasia) Normal (compared with host adjacentcartilage) 0 Slightly reduced 1 Significantly reduced 2 No metachromaticstain 3 Surface regularity Smooth (>¾^(a)) 0 Moderate (½ < ¾^(a)) 1Irregular (¼ < ½^(a)) 2 Severely irregular (<¼^(a)) 3 Thickness of thecartilage 0 > ⅔^(b) 0 ⅓ > ⅔^(b) 1 <⅓^(b) 2 Integration of donor withhost adjacent cartilage Both edges integrated 0 One edge integrated 1Neither edge integrated 2 Total maximum 14 ^(a)Total smooth area ofreparative cartilage compared with the whole area of the cartilagedefect. ^(b)Average thickness of reparative cartilage compared with thatof surrounding cartilage.

Statistical Analysis

Statistical significance was evaluated by using t-test. The significancelevel was p<0.05.

Results

Costal Chondrocytes Cultured in Sponge-form Scaffolds

The proliferation rates of costal chondrocytes in three differentscaffolds; collagen-GAG sponge (COL-GAG), chitosan sponge (CS), andHA-coated chitosan sponge (CS-HA), were compared at 1, 2 and 4 weeks ofculture using MTT assay (FIG. 17).

The value in FIG. 17 shows cell proliferation inside these scaffolds.The O.D. value of the CS and CS-HA were gradually increased with theculture time and there was no notable difference between two scaffolds.Similar trend was found in COL-GAG, but initially started low level. At28 day culture, the O.D. values compared with that at 7 day culture,were increased approximately 1.30, 1.43 and 1.40 folds in COL-GAG, CSand CS-HA, respectively.

To estimate neosynthesis of collagen of the cell seeded compositescaffolds, procollagen Type I C-peptide (PIP) was measured by ELISAassay (FIG. 18). At 7 day culture, collagen syntheses of CS and CS-HAwere more than that of COL-GAG with significant difference. However, at14 days, collagen synthesis in CS was more extensive than in COL-GAG andCS-HA (CS>CS-HA>COL-GAG). Compared with at 7 day culture, collagensynthesis at 14 day culture was significantly decreased. Duringfour-week culture period, in CS, collagen secretion to culture mediumwas gradually decreased, on the contrary, in COL-GAG and CS-HA, collagenrelease were significantly decreased between 1 and 2 weeks, and thenincreased between 2 and 4 weeks.

Blyscan assay revealed that the amounts of GAGs released from the costalchondrocytes-seeded three different scaffolds into culture supernatantfor a week (FIG. 18). At the 1 week of culture, both chitosan scaffoldsreleased more GAGs than COL-GAG. At the 14 days, the released amounts ofGAGs were similar in all three scaffolds, but at the 28 days of culture,the relseased GAG amounts in COL-GAG were very smaller than in CS andCS-HA.

During rabbit costal chondrocytes were seeded onto COL-GAG, CS and CS-HAand cultured for 28 days, CS and CS-HA were bigger in size than COL-GAGthroughout the culture time.

Histological examination of these specimens using H & E stains revealedmorphologic characteristics (FIG. 19). In FIG. 19, S represents surface,C represents center, scale bar represents 50 μm, black arrow representsfibrous tissue, white arrow represents death cell, black arrow headrepresents round-shaped cell, and white arrow head representsspindle-shaped cell. After 2 day chondrocytes culture, the seeded cellswere mainly presented in the superficial area of both chitosan-basedscaffolds and especially COL-GAG. Round and spindle-shaped cells weremixed and no cell was occupied in lacunae at 2 days. The cells in thecenter of the CS and CS-HA were aggregated but in the COL-GAG, the cellsin the center were rare and dispersed at 2-day specimen.

After 7 days of culturing, the increase of the cell number was notablein the periphery of the scaffolds, and new cell peripheral matricessurrounded most of the cells. In the center and the periphery of both CSand CS-HA scaffolds, the vast majority of the seeded cells hadmaintained their spherical morphology, occupied in lacunae, andsynthesized abundant matrices. However, in COL-GAG, the cells of theperiphery were round morphology, but they were not occupied in lacunae,and the cells in the center were spindle-shape morphology and were notaggregated.

At 14 day of culture, the vast majority of the cells in the center andthe periphery of both chitosan-based scaffolds had still maintainedtheir spherical morphology, occupied in lacunae, and synthesized muchabundant extracellular matrices. But in COL-GAG, cells of the peripherywere spherical morphology, occupied in lacunae, and synthesized abundantextracellular matrices, but cells of the center were stillspindle-shaped, had small lacunae, and synthesized a little ECM.

At the end of the experiment (28 days), the surface of bothchitosan-based scaffolds was covered with thin fibrous tissue. The cellsin the center as well as in the periphery became small, were stilloccupied in large lacunae. In COL-GAG, the cells in the peripherydecreased cellularity, had very small round morpholohgy, and did nothave lacunae. The cells in the center were similarly at 14 days.

Histological staining for cartilaginous ECM of GAG by Safranin-O fastgreen (S/O) staining revealed that GAG was detected in the CS and CS-HAscaffolds in the center of the scaffolds throughout the period ofculture (FIG. 20). In FIG. 20, white arrow head represents Safranin-Ostained frame, black arrow represents fibrous tissue, white arrowrepresents fast green stained cells, and scale bar represents 100 μm. Inboth chitosan-based scaffolds, cells in the center were stained with S/Ofrom 2 day culture specimens. However, in COL-GAG, cells in the centerwere not stained with S/O. The amount of accumulated GAG increased withan increase of the culture time in the three types of scaffolds. At 28day culture, the surrounds of the scaffolds were not stained with fastgreen.

No GAG was histological detected in the CS and CS-HA withoutchondrocytes, but the frame of COL-GAG was positively stained with S/Obecause they conjugated with chondroitin sulfate.

Immunostaining for Type I and Type II Collagen

In both chitosan-based scaffolds, type I collagen was not detected in 1and 2 week specimens. On the contrary, anti-type II collagen antibodywas strongly positive in both chitosan-based scaffolds during 1 to 2weeks culture period. At 4 weeks, some portions of cell aggregates inthe surface and center of the scaffolds were positive with anti-type Icollagen antibody. However, in the 4 week specimen, anti-type IIcollagen antibody was still positive, but became weak.

In COL-GAG, type I collagen was detected in all 1, 2 and 4 weekspecimens. Anti-type II collagen antibody was positive in only thesurface at 1 week, and in the matrices of the periphery and a few ofcells in the center at 2 weeks. In the 4 week specimen, anti-type IIcollagen antibody was very weakly positive (FIGS. 21 and 22). In FIG.21, black arrow represents fibrous tissue, black arrow head representstype I collagen expressing cell, white arrow represents COL-GAG frame,and scale bar in FIGS. 21 and 22 represents 100 μm.

As a result of the histological observation above, when dedifferentiatedchondrocytes were seeded onto three types of sponge form scaffolds (CS,CS-HA and COL-GAG), in both chitosan-based scaffolds, the seeded cellswere redifferentiated in morphology, GAG synthesis and type II collagenexpression for 1 to 2 weeks. However, in COL-GAG, internal cell seedingwas not effective, and redifferentiation of cells in the center was notgood. Very lately, at 4 weeks, cells began to lose synthesized ECMactivity (the size of lacunae became smaller).

In vivo Results

This study was designed to evaluate the role of cultured costalchondrocytes-chitosan composite in the repair of full-thicknessarticular cartilage defects in a weight-bearing animal model.

Costal chondrocytes at P2 were seeded onto CS-HA scaffolds, and culturedfor 2 weeks. Cartilage defects were surgically made on rabbit patellagroove of the femur. Animals were euthanized, and the knee joints wereobtained at 6 and 12 weeks. The specimens were analyzed macroscopically,histologically and immunohistochemically.

Clinical Test

No postoperative complications occurred in any of the experimentalanimals. During the study, no symptom of articular outflow anddisability was observed.

Gross Appearance

Upon gross observation, no symptom of synovial membrane outflow orsynovitis was observed in any joint. Although synovial tissue histologywas not performed, there was no gross evidence of an inflammatoryreaction. Grossly, the synovial fluid in all joints was normal in termsof quantity, viscosity, and color.

In FIG. 23 to FIG. 26, Control represents the control group, Srepresents CS-HA grafted defect, and S-CELL represents costalchondrocytes-seeded CS-HA grafted defect.

Empty (Control) Defect

At 6 weeks after transplantation, in the control group, the defects wereseen smooth surface. The defects had a gap or lack of continuity betweenthe repair tissue and the adjacent articular cartilage. The margins ofthe defects were discernable. The repair tissue was whiter and slightlyopaque compared with the adjacent normal cartilage (FIG. 23). At 12weeks after transplantation, the repair tissues in the control groupwere easily distinguished from the normal adjacent cartilage becausethey had white and opaque appearance. The defects were completely filledwith smooth white repair tissue (FIG. 23).

CS-HA Grafted Defect

Grossly at 6 weeks, some of the defects were not completely filled withrepair tissue, and in some areas subchondral bone was exposed. Thesurface of the repair tissue in the defects was more irregular than thatsurrounding normal, and the margins of the repair tissue were notsmooth. The repair tissues were white to pink appearance andtranslucence (FIG. 23). At 12 weeks, the repair tissues of CS-HA grafteddefects were still discernible, but were hyaline in appearance andtexture. And, the defects were filled to the level of the adjacentnormal cartilage. The surface of some of them was smoother than that of6-week specimen. In some cases (1/8), the repair tissues were filledwith not cartilage, but bone (FIG. 23).

Costal Chondrocytes-laden CS-HA Grafted Defect

At 6 weeks, all the defects were filled with repair tissues, and themargins of the defects were discernable from the adjacent normalcartilage. The surface and the edges of the repair tissue were rough andirregular. The repair tissue was hyaline in appearance and texture (FIG.23). Grossly at 12 weeks, all the repair tissues formed the level of thesurrounding normal cartilage. The surface and the edges of the repairtissue were smoother than that of 6-week specimen. The repair tissuecould still be distinguished from the surrounding normal cartilage. Butthe edges of the repair cartilage were more smooth integrated with thenormal cartilage than that in 6-week specimen (FIG. 23).

At 6 weeks, gross appearance of CS grafted defects was similar to thatof cells-laden CS grafted defects: both of them were white and somehowirregular. Up to 12 weeks, in general, the surface of the controldefects was much smoother than that of CS grafted defects wherein thesurface is protruded and irregular. The surface of the control defectswas white appearance, but that of grafted defects was pink appearance.The color and texture of the repair tissue in cell-laden grafted defectswere similar to those of the adjacent normal cartilage.

Morphology

Control Defect

At 6 weeks after transplantation, addition resembling fibrocartilagioustissue formation was seen at the graft copula. The defects had a gap orlack of continuity between the repair tissue and the adjacent articularcartilage on one or both sides. This observation indicates decrease inintegration between the surrounding articular artilage and the repairtissue. The interface between the repair cartilage and the adjacentnormal cartilage occasionally showed fibrillar continuity. The surfaceof the repair tissue was composed with 3 to 5 layer of flattened celland was fibrillation. The repair tissue consisted of a mixture offibrous tissue and hypercellular fibrocartilage. Some defects showedcracked gap in fiber and fibrocartilagious reparative cartilage. Therepair tissue recovered subchondral bone in good degree (FIGS. 24 to26).

At 12 weeks, some of the cartilage defects had a gap or lack ofcontinuity between the repair tissue and the adjacent normal cartilage.The surface of the repair tissue was smooth and well fibrillation. Thedefects were filled with chondrocytes and matrices. However, fibroblastsare predominant in the surface layer, and most of the surface wereconsisted of fibrous matrices. The repair tissues had a fine ECMresembling fibrous cartilage and were involved with round, relativelymature chondrocyte-like cell. So the repair tissues of the control groupat 12 weeks were consisted predominantly of fibrous tissue andfibrocartilagious tissue (FIGS. 24 to 26).

CS-HA Scaffold Grafted Defect

At 6 weeks, the repair tissues were good integration with the adjacentnormal cartilage. In microscopic view, the repair tissues werehypercellular components. The repair tissues were predominantlyhypertrophic chondrocytes, and flattened superficial cells inosteochondral defects treated with chitosan-based sponge. The bottom ofthe repair tissue was composed spindle-shaped cells which have irregularECM and are similar to immature substantial cell. The repair tissue washyaline and fibrous cartilage. A few cell layers on the surface of thedefects were small and flattened, and ECM was stained with H & E. Almostall the fibrous tissues were found at the surface of the repair tissue.The middle layer of the repair tissue had hyaline cartilage like tissue,which involved with round and large nucleus, relatively immaturechondrocyte-like cell (FIGS. 24 to 26). However, the repair cartilagewas seen perpendicular crack, but not very often.

At 12 weeks, the repair tissue and the adjacent normal cartilage wereintegrated well. After 12 weeks of transplantation, the repair tissuehad predominantly immature chondrocytes and flattened superficial cellsin osteochondral defects treated with chitosan-based sponge. Theelongated fibroblasts were aligned parallel to the surface and thedefect was filled with high density immature chondrocytes andhyaline-like matrices. The bottom of the repair cartilage involvedhypertrophic chondrocytes and well restored to subchondral bone.Occasionally, some of the defects were filled with not cartilaginoustissue but osseous tissue that covered with thin fibrous tissue (FIGS.24 to 26).

Costal Chondrocytes-laden CS-HA Scaffold Grafted Defect

In the 6 week specimens, chondrocytes in the reparative tissue weregreater numbers than in adjacent normal cartilage, and they werecolumnar organized. Some part of the surface was evidence offibrillation. The repair cartilage showed a normally integration withthe surrounding normal cartilage and the bony portion. Any defectsshowed cracked gap in the repair cartilage (FIGS. 24 to 26). Bonetrabecules developed in the deep zone of the graft, enclosing smallresidue chondrocyte-island was seen in the cancellous bone.

At 12 weeks, the defects treated with the cultured cell-laden CS-HA werecompletely filled with cells and matrix. The repair tissue was normallyintegration with subchondral bone and adjacent normal cartilage. Anydefects showed cracked gap in the repair cartilage. The reparativetissue involved with single or multiple chondrocytes within singlelacunae and they consisted isogenous groups and were columnar arranged.The repair tissue had abundant extracellular matrix and orderly alignedchondrocytes, and no detect had fibrillation of the surface althoughslight rough surface was seen. The chondrocytes of 2 to 4 layers in thesurface were small and flattened-shape, and involved lacunae. Thechondrocytes in the bottom of the repair tissue were large and roundcells and hypertrophied, larger number than them in the surface. Therepair cartilage was predominantly hyaline cartilage. The thickness ofthe hyaline cartilage formed in these defects was thinner than that ofthe adjacent normal cartilage (FIGS. 24 to 26).

GAG Distribution—Safranin-O Staining

Control Defect

At 6 weeks, in the control defects, only some area in the bottom of therepair tissue was stained with S/O. The other repair tissue was notstained with S/O, and the surface of the repair tissue was stained withfast green, opposite staining color. A moderate to severe loss ofmetachromasia was apparent in the control defect sections (FIG. 27). At12 weeks, the surface of the repair tissue was still stained with fastgreen. Only in one case, the repair tissue was stained with S/O, and theother cases were negative stain for S/O (FIG. 27).

CS-HA Scaffold Grafted Defect

At 6 weeks, the areas filled with hypertrophied chondrocytes werepositive stain with S/O, and the adjacencies to the surrounding normalcartilage were weakly stained. Safranin-O and fast green staining wasevident predominantly in the area of hypertrophic chondrocytes at thebottom of the defects (FIG. 27). At 12 weeks, the bottom of thereparative tissue and the adjacencies to the surrounding normalcartilage were more strongly stained with S/O than that at 6 weeks (FIG.27).

Costal Chondrocytes-laden CS-HA Scaffold Grafted Defect

At 6 weeks, all of the repair tissue were stained with S/O but slightlyor moderately decreased as compared to the surrounding normal cartilage,and the bottom of the repair tissue was strongly positive for S/O. Thestaining intensity of the repair cartilage decreased weakly ormoderately as compared to the adjacent normal cartilage (FIG. 27). At 12weeks, the repair tissue was stained with S/O to the level of thesurrounding normal cartilage except some areas in the center. Most ofthe repair cartilage was seen hyaline-like, and the defect showedperpendicular columnar chondrocytes in the radius area.

Immunostaining for Type I and Type II Collagen

Control Defect

At 6 weeks after transplantation, half of the upper portion of therepair tissue and the adjacencies to the normal cartilage were positivewith anti-type I collagen. The other portions except the calcifiedcartilage were positive with anti-type II collagen (FIG. 28). At 12weeks, the cells in whole portions of the repair tissue were positivewith anti-type I collagen (FIG. 28).

CS-HA Grafted Defect

At 6 weeks, the edges and upper portion of the repair tissue werepositive with anti-type I collagen. The hypertrophied portion was alsopositive with anti-type I collagen (FIG. 28). At 12 weeks, the surfaceof the repair tissue was positive with anti-type I collagen, and theothers were anti-type II collagen (FIG. 28).

Costal Chondrocytes-laden CS-HA Scaffold Grafted Defect

At 6 weeks, some portions of the surface of the repair tissue werepositive with anti-type I collagen. Anti-type II collagen expression wasreduced in the repair tissue compared to the adjacent normal cartilage(FIG. 28). At 12 weeks, in the small portion of the surface, anti-type Icollagen was slightly positive expression. The repair tissue expressedanti-type II collagen in a similar level to the normal cartilage (FIG.28).

The results of the histological grading scale (mean score) was shown inTable 5.

TABLE 5 Results of the histological grading scale Control S S-CELL 6 w12 w 6 w 12 w 6 w 12 w Cell morphology  1.67 ± 0.58^(a) 2.5 ± 0.84 2.25± 0.96 2.5 ± 1.2 1.75 ± 0.96  1 ± 0.71 Matrix stain 2 ± 0 2.5 ± 0.84 2.5 ± 0.58 2.13 ± 0.83   2 ± 0.82 0.8 ± 0.45 Surface regularity 0.33 ±0.58 1.17 ± 0.75  0.5 ± 1    1 ± 0.76 0.75 ± 0.5  0.4 ± 0.55 Thicknessof 0 ± 0 0.5 ± 0.84 1.25 ± 0.96 1.25 ± 0.89 0.75 ± 0.96 0.8 ± 0.45cartilage Integration with host 1 ± 0 0.67 ± 0.52  0 ± 0 0.13 ± 0.35 0 ±0 0.2 ± 0.45 adjacent cartilage Total 5 ± 1 7.33 ± 2.4   6.5 ± 3.11   7± 2.62 5.25 ± 2.99 3.2 ± 1.79 ^(a)represented by S.D.

Example 6: Evaluation of Repair Effect of MSC-like DedifferentiatedCells within Chitosan-based Scaffold on Articular Cartilage DefectsMaterials and Methods

Isolation and Culture of Chondrocytes

As the Example 1, costal chondrocytes were isolated from costa by enzymetreatment, plated at a cell density of 5×10⁵ cells/100 mm diameter Petridish, and subcultured in MSCGM added with 1 ng/ml of FGF up to P8 as theExample 3, to obtain MSC-like dedifferentiated cells.

Preparation of MSC-like Dedifferentiated Cells within Chitosan-basedScaffolds and Culture

As the Example 5, chitosan sponge was prepared and coated with HA. TheCS-HA sponges in 5 mm diameter were seeded with 2×106 MSC-likededifferentiated cells at P8 to obtain MSC-like dedifferentiated cellswithin scaffolds, and cultured in chondrogenic medium containingTGF-beta for 2 weeks to induce into cartilage differentiation. For theestimation of the degree of differentiation into cartilage, Safranin-Ostaning was performed.

Repair Effect on Articular Cartilage Defect

As the Example 1, the rabbits were anesthetized, and administeredantibiotics prior to operation. Defects (5 mm diameter, approximately2.0 to 2.5 mm depth) were made on the patellar grooves of the bothfemur, and grafted with MSC-like dedifferentiated cells (alreadydifferentiated into cartilage) within scaffold as the Example 2.

Results

Redifferentiation into Chondrocytes

FIG. 29 is a macroscopic picture of the artificial cartilage containingredifferentiated chondrocytes obtained by loading MSC-likededifferentiated cells from costal chondrocytes onto chitosan sponge andredifferentiating them in chondrogenic medium.

FIG. 30 shows the results of Safranin-O staining for GAG in chondrocytesredifferentiated in chondrogenic imdium. After 2 weeks of chondrogenicdifferentiation, MSC-like dedifferentiated cells in the sponge wereredifferentiated into chondrocytes.

Articular Cartilage Defect Repair

FIG. 31 is a macroscopic picture of rabbit articular cartilage defect at6 weeks after transplantation with MSC-like dedifferentiated cells ladenchitosan-based scaffolds. The repair tissue was smoothly integrated withthe adjacent normal tissue, and was seen hyaline cartilage in appearanceand texture.

INDUSTRIAL APPLICABILITY

In the present invention, it was first disclosed that costal cartilageprovides higher cell yield and cell expansion rate than articularcartilage, and so costal cartilage is better cell source for cartilagerepair than articular cartilage. Before the present invention, it wasknown in the art that dedifferentiation rate, that is, loss ofchondrocytic phenotype during the culture of chondrocytes wassignificantly high which limits ACT application. However, surprisingly,in the present invention, it was confirmed that dedifferentiatedchondrocytes during the passage of costal chondrocytes obtained fromcostal cartilage show MSC properties, and so such fully dedifferentiatedcostal chondrocytes can be differentiated into desired differentiatedcells such as osteoblasts, adipocytes, etc, as well as chondrocytes whenrecultured in differentiated condition. Thus, the present inventionprovides an artificial cartilage and cell therapeutic agent containingMSC-like dedifferentiated cells obtained by passaging costalchondrocytes.

In addition, the present invention showed that autologous costalchondrocytes-loaded chitosan-based scaffold when transplanted intocartilage defect can very effectively repair full-thickness articularcartilage defects in a weight-bearing site.

What is claimed is:
 1. A process for preparing an artificial cartilagecomprising: passaging costal chondrocytes in fibroblast growth factor(FGF)-containing mesenchymal stem cell growth medium (MSCGM) to obtainMSC-like dedifferentiated cells; and obtaining the artificial cartilage,wherein the dedifferentiated cells exhibit a fibroblastic spindle shape,MSC properties and an increased expression of cartilaginousextracellular matrix when redifferentiated into cartilage.
 2. Theprocess of claim 1, which further comprises culturing the MSC-likededifferentiated cells in a chondrogenic medium to obtainredifferentiated chondrocytes.
 3. The process of claim 2, which furthercomprises pellet culturing the MSC-like dedifferentiated cells in achondrogenic medium to obtain redifferentiated chondrocytes.
 4. Theprocess of claim 1, which further comprises loading the MSC-likededifferentiated cells on a chitosan-based scaffold.
 5. The process ofclaim 1, which further comprises loading the MSC-like dedifferentiatedcells on a chitosan based scaffold and passaging costal chondrocytes infibroblast growth factor (FGF)-containing mesenchymal stem cell growthmedium (MSCGM) from passage 4 to passage 8.